In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Summary

In the present protocol, the endothelial barrier function of the submandibular gland (SMG) was evaluated by injecting different molecular weighted fluorescent tracers into the angular veins of test animal models in vivo under a two-photon laser-scanning microscope.

Abstract

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the endothelial barrier dysfunction is related to many salivary gland secretory disorders. The present protocol describes an in vivo paracellular permeability detection method to evaluate the function of endothelial tight junctions (TJs) in mouse submandibular glands (SMG). First, fluorescence-labeled dextrans with different molecular weights (4 kDa, 40 kDa, or 70 kDa) were injected into the angular veins of mice. Afterward, the unilateral SMG was dissected and fixed in the customized holder under a two-photon laser-scanning microscope, and then images were captured for blood vessels, acini, and ducts. Utilizing this method, the real-time dynamic leakage of the different-sized tracers from blood vessels into the basal sides of the acini and even across the acinar epithelia into the ducts was monitored to evaluate the alteration of the endothelial barrier function under physiological or pathophysiological conditions.

Introduction

Various salivary glands produce saliva, which primarily acts as the first line of defense against infections and helps digestion, thereby playing an essential role in oral and overall health¹. Blood supply is crucial for salivary gland secretion since it constantly provides water, electrolytes, and molecules that form the primary saliva. Endothelial barrier function, regulated by the tight junction (TJ) complex, strictly and delicately limits the permeation of capillaries, which are highly permeable to water, solutes, proteins, and even cells moving from the circulating blood vessels into the salivary gland tissues^2,3. We have previously found that the opening of the endothelial TJs in response to a cholinergic stimulus facilitates saliva secretion, whereas the impairment of the endothelial barrier function is interlinked with hyposecretion and lymphocytic infiltration in the submandibular glands (SMGs) in Sjögren's syndrome⁴. These data suggest that the contribution of endothelial barrier function needs to be paid enough attention regarding a variety of salivary gland diseases.

A two-photon laser-scanning microscope is a powerful tool for observing the dynamics of cells in intact tissue in vivo. One of the advantages of this technique is that near-infrared light (NIR) has deeper tissue penetration than visible or ultraviolet light when specimens are excited by NIR and does not cause obvious light damage to tissues under appropriate conditions^5,6. Indeed, the salivary glands are a very homogenous and superficial tissue, in which the surface acinar cells are only around 30 µm away from the gland surface^7,8. It has been shown that intravital confocal microscopy can study exocrine secretion and actin cytoskeleton in live mouse salivary glands at subcellular resolution⁸. Two-photon laser-scanning microscopy, nevertheless, not only has the advantage of conventional confocal microscopy but can also be used to detect deeper tissue and image more clearly. Here, fluorescence-labeled dextrans, which are frequently used as paracellular permeability tracers and have the advantage of different sizes, can be used to test the magnitude of TJ pore⁹. In the present study, an intravital real-time two-photon laser-scanning microscopy technique is established for in situ evaluation of endothelial barrier function in mouse SMGs. Each work step for in vivo vascular permeability detection in mouse SMGs is described in the current protocol. Here is an example of detecting endothelial barrier function in the mouse SMG duct ligation model.

Protocol

All experimental procedures were approved by the Ethics Committee of Animal Research, Peking University Health Science Center, and complied with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). Male wild-type (WT) mice in the age group of 8-10 weeks were used for the present study. The experimental animals were carefully treated to minimize their pain and discomfort.

1. Animal procedures

1. Prepare and administer the anesthetics and tracers.
   1. Maintain sterile conditions throughout the study and sterilize the instruments by autoclaving. Divide the mice into two groups.
      NOTE: The control group was left untreated, and the other group with unilateral SMG duct ligation for 1 day served as the experimental group. When grouping, mice with similar body weight and age need to be selected to reduce the influence of weight and age on vascular permeability.
   2. Choose appropriate tracers such as fluorescein isothiocyanate (FITC)-labeled dextran (molecular weight: 4 kDa; FD4) and rhodamine B-labeled dextran (molecular weight: 70 kDa; RD70) (see Table of Materials) with distinct excitation/emission spectra to minimize the interference between fluorescence signals (using the FITC or rhodamine B filter, respectively) for the permeability assay.
      NOTE: The excitation wavelengths of FD4 and RD70 are 488 nm and 594 nm, respectively, and the selected emission wavelengths are 511-564 nm and 617-669 nm, respectively.
   3. Dilute the tracers in sterile phosphate-buffered saline (1x PBS) into 100 mg/mL stocks and store the aliquots protected from light at −20 °C.
      NOTE: Dextran with a molecular weight such as 4 kDa will rapidly leak from the blood in mouse SMGs even without pathological conditions¹⁰.
   4. Intraperitoneally inject the tribromoethanol (the dose for a 25 g mouse was about 600 µL) to anesthetize the mice. Provide analgesia as recommended by the local animal ethics committee.
      NOTE: After the induction of anesthesia, use vet ointment on the eyes to prevent dryness. Take good care of the animals¹¹. Wait until the mouse tolerates mechanical stimulation e.g. toe pinch without motor response.
   5. Inject a mixture of two paracellular permeability tracers with different molecular weights (FD4 and RD70) into the angular vein (0.5 mg/g body weight). Inject each mouse with a 100 µL tracer solution mixture, with 50 µL of each dye mixed in a 1:1 ratio.
      1. After anesthesia, gently hold the head and neck of the mouse to make one side of the eyeball protrudes slightly. Insert an insulin syringe containing fluorescent tracers (be careful not to introduce any air into the syringe) along the corner of the eye at a right angle to the eye.
         ​NOTE: If the angular vein is inserted correctly, the dye solution will be easily injected into the vein, and there will be no resistance during the injection. Additionally, tail vein injection in mice might also be a candidate for intravenous tracer molecules.
2. Perform the SMG isolation following the steps below.
   NOTE: Use sterile tools, including tissue scissors and separation nickels, for the surgery.
   1. Separate a unilateral gland carefully so as not to damage the surrounding blood vessels and nerves under a stereoscopic microscope.
      1. After the injection of paracellular permeability tracers, quickly put the mice on the cardboard and place them in a supine position with their limbs and heads taped. Apply hair removal cream to the neck skin to remove the mouse's hair. Use 75% ethanol to disinfect the skin before surgery.
      2. Then, under a stereomicroscope, cut the epidermis of the neck with general tissue scissors to expose both sides of the SMGs (the right gland was treated in this experiment). Next, use a blunt tissue separation nickel (see Table of Materials) to gently separate the capsule from the gland surface, taking care not to damage the gland tissue and blood vessels.
      3. Separate the capsule from the gland surface without damaging the glandular structure. In addition, ensure that the entire process of mouse restraint and gland separation is accomplished within 10 min.

2. Two-photon microscope set-up

1. Connect the customized holder to the negative pressure device, and check whether the negative pressure effect is properly achieved in advance.
   NOTE: The holder is shaped like a miniature magnifying glass with a flat piece of round glass in the center (diameter: 4.32 mm, Figure 1). The negative pressure device was designed in-house in such a way that the holder is connected to a rubber tube, which is linked with a drainage bottle, and the bottle is attached to the negative pressure controller through a short rubber tube. In this way, the negative pressure controller can adjust the pressure to ensure that the tissue is sucked up into the holder.
   1. Link one side of the holder with the negative pressure device. To test whether the negative pressure device is intact and appropriate, turn the holder upside down, add a drop of water, and set the negative pressure value to 20 units. If the water is completely and quickly sucked away, the negative pressure device is successfully installed with the holder.
2. Gently place the exposed SMG of the mouse on the customized holder, and suck up the tissue by negative pressure suction as far away from the mouse body as possible to reduce motion artifacts caused by respiration and other conditions.
3. Image the support material attached to the gland under a 25x/NA 1.0 water immersion objective (special for NIR).
   ​NOTE: Use an upright two-photon microscope (see Table of Materials) in this experiment. The blood vessels must be visible immediately with obvious fluorescence after the tracer injection.

3. Vessel imaging and permeability detection

1. Commence imaging soon after the sight of the gland was chosen.
   NOTE: Typically, time series of 45-50 images in 20 s or longer are generally captured depending on the experimental design.
2. Acquire sequential images along the Z-axis, stepped 2 µm apart, from the gland's surface up to 70-140 µm into the tissue to generate a three-dimensional (3D) construction.
3. Acquire time-lapse imaging of the vessels to measure vascular permeability in the SMG.
   NOTE: Set the conditions of the program menu as follows after running the microscope software (see Table of Materials).
   1. In the Explorer dialog box, click on Add New Folder and change the file name.
   2. Go back to the Acquisition column. In XY, the Format is 512 x 512, and the Speed is 400 Hz. Turn Bidirectional X on.
   3. Set the Zoom factor to 0.75 and 1.5 for Zoom.
   4. To acquire time-lapse imaging, select xyt under Acquisition Mode. Set Duration to 20 s, 30 min, or longer according to the experimental need.
   5. To make the 3D construction, set the Acquisition Mode to xyz. Z-Step size can be set according to the actual situation.
   6. After setting the conditions, click on Live to observe the vascular imaging of two channels and overlapping channels in the photo forming frame and choose the areas of interest.
   7. Click on Start to start imaging.
      ​NOTE: The mouse must not be left unattended during the imaging process while under anesthesia. 

4. Data analysis

1. To evaluate the vessel permeability, use Image J software to calculate the fluorescence intensity ratio of FD4 and RD70 between the outside and inside vessels and denote them as extra- and intravascular intensity⁴.
   NOTE: The alteration of vascular permeability through tracing the transport of dextrans reflects the change in endothelial barrier function. Moreover, observe the vascular morphology and density by calculating the diameter and number of vessel changes.
   1. Run Image J software (see Table of Materials).
   2. Click on File and select Open to open the blood vessel images captured under the two-photon microscope.
   3. Select Image and set Type to 16-bit to convert the image format.
   4. Select Analyze and select Area, Mean, IntDen under Set Measurements. Click on OK to confirm.
   5. Under Analyze, select Tools, open ROI Manger, and select Show All and Labels.
   6. Measure the intravascular fluorescence intensity value: click on the lasso tool on the main interface to sketch the outline of the blood vessel. Click on Add in ROI Manager. Follow this step to continue to select multiple vessels. Select all of the ROI Manager's objects and click on Measure.
   7. Measure the total fluorescence intensity value: outline the whole image, click Measure, and the result is the total fluorescence intensity value of the image.
   8. Calculate the extravascular fluorescence intensity value. Copy the data to Excel for calculation. Extravascular fluorescence intensity ratio = (1 − intravascular fluorescence intensity value/total fluorescence intensity value) × 100%.

5. Downstream applications

1. During the experiment, calculate the blood flow rate by measuring the distance that a red cell (shown as a black dot under the two-photon microscope)⁴ undergoes in a time duration.
2. To visualize the movement of blood cells across the endothelial cells, label the blood cells by fluorescence. Then, inject the fluorescence-labeled cells with FD4 or RD70 via the mouse tail vein. After circulating for 5 min, trace the interested cells for a period.
   ​NOTE: A total of 2 x 10⁶ CFSE (carboxyfluorescein succinimidyl ester, a membrane-permeable fluorescent dye)-labeled lymphocytes derived from the spleen of donor mice were transferred into recipients by tail vein injection, and the infiltration of lymphocytes into SMGs was investigated in experimental animal models⁴.

6. Animal care and recovery

1. Take good care of the animals throughout the experiment.
   NOTE: Ensure that the animal that has undergone surgery is not returned to the company of other animals until fully recovered during the experiment.
2. Do not leave the animal unattended until it regains enough consciousness.
   NOTE: Observe the breathing status of the mice continuously and keep them warm by putting them on the animal heating pad. Provide postsurgical pain recovery housing and analgesia, as recommended by the local animal ethics committee.

Representative Results

Following the protocol, the unilateral SMG was attached to a custom-made holder, and the gland was kept as far away from the mouse body as possible to prevent breathing from causing motion artifacts. The rapid flow of the red blood cells (black dots) in blood vessels was observed under the microscope. After finding the tissue field under an ocular lens, one must switch to manipulate the microscope software. In the control group, both the tracers existed in the blood vessels of the mouse SMG. In particular, due to its small molecular weight, FD4 was able to leak out of the blood vessels to the basal sides of acini and ducts, thereby clearly depicting the shape of the acini and ducts (as A and D point out in Figure 2A). By contrast, dextrans with higher molecular weights, such as 40 kDa and 70 kDa, could not differentiate the SMG morphology. Indeed, RD70 was dominantly distributed in large-sized blood vessels and microvessels. In the duct ligation group, both FD4 and RD70 were extravasated to the basal sides of acini, which indicated that duct ligation could disrupt the endothelial barrier function and then increase the permeability to large molecules. The semi-quantitative FD4 and RD70 fluorescence intensity results also confirmed the above phenomena (Figure 2B). Besides, the diameter of the blood vessels was increased, indicating that duct ligation for 1 day induced dilation of blood vessels. Furthermore, the 3D images showed much more obscured fluorescence of FD4 and RD70 around blood vessels in the ligation group (Figure 2C).

Figure 1: A schematic diagram showing the customized holder. The holder is shaped like a miniature magnifying glass with a flat piece of round glass in the center (diameter: 4.32 mm). One side of the holder is connected with the negative pressure device to suck up the tissues under the glass, while the other side of the holder is dead. Please click here to view a larger version of this figure.

Figure 2: In vivo vascular permeability assay and 3D images of blood vessels in mice submandibular glands (SMGs). The mice were divided into control and duct ligation groups. The unilateral glands in both groups were exposed and observed. (A) The in vivo vascular permeability assay was performed by injecting 4 kDa FITC-labeled dextran (FD4) and 70 kDa rhodamine B-labeled dextran (RD70) into the angular vein. Arrowheads indicate the changes in the distribution of tracers in the SMG. A, acini. D, duct. Scale bar = 100 µm. (B) For the semi-quantification analysis of the above images, the fluorescence intensity, including FD4 and RD70 within blood vessels (intravascular) and outside blood vessels (extravascular), was measured by the Image J software. (C) 3D images of blood vessels and microvessels in SMG. Arrowheads indicate the changes in the distribution of tracers in the SMG. Please click here to view a larger version of this figure.

Discussion

The maintenance and regulation of endothelial barrier function are essential for vascular homeostasis. Endothelial cells and their intercellular junctions play a critical role in maintaining and controlling vascular integrity¹². The shear force of blood flow, growth factors, and inflammatory factors can cause changes in vascular permeability and, thus, participate in the occurrence and development of systemic diseases such as hypertension, diabetes, and autoimmune diseases^13,14,15. The vascular distribution and blood flow of the salivary gland is one of the most abundant in all organs and tissues, but research on the vascular system of the salivary gland has not been carried out widely. A deeper and more comprehensive understanding of salivary gland blood vessels, particularly the endothelial barrier function, will provide new clues to the mechanism of salivary secretion and promote vascular biology research.

The in vivo two-photon laser-scanning microscopy technique can quantify the vascular permeability by intravenous injection of fluorescent dye without sacrificing the animal. Fluorescence-labeled dextrans with microspheres of different molecular weights or sizes can better assess the extent of endothelial injury. Meanwhile, the dynamic kinetics of vascular permeability are determined in real-time by using the intravital evaluation system of vascular permeability. Another advantage is that it is easy to distinguish the types of blood vessels, such as arterioles, venules, and capillaries¹⁶. Furthermore, destruction of the vascular endothelial barrier and migration of immune cells are inevitably linked to inflammation, but there are many studies investigating a single factor, and only a few studies have focused on the connection between both aspects^17,18. Uhl et al. recently established a research method to analyze the role of different pathogenic factors in salivary gland diseases by injecting fluorescence-labeled dextrans and immune cell-specific antibodies with different fluorescent markers simultaneously in vivo¹⁹. Here, fluorescence-labeled immune cells can also be traced through tail vein injection to explore pathogenesis in the current working model. Therefore, the present experimental method may provide a unique system for studying the interaction between leukocyte extravasation and endothelial barrier integrity in SMG disease animal models in vivo.

Nevertheless, it must not be ignored that the responsiveness of mice to anesthetics is different, and the longer the imaging time takes, the longer the anesthesia time needed, and the more difficult it is for mice to recover. Therefore, this experiment is better used to investigate when no subsequent in vivo experiments are required after the imaging. In addition, another limitation is that this technique is not suitable for experiments with a long time span. Dextrans are water-soluble and easy to metabolize, resulting in weaker fluorescence with a longer imaging time, and thus, it is better to maintain imaging time within 30 min.

Altogether, the study focuses on the penetration of paracellular permeability tracers and cells from blood vessels and microvessels into glandular tissues and has rigorously established contrast-enhanced intravital dynamic two-photon laser-scanning microscopy as an advanced method to measure vascular permeability to evaluate endothelial barrier function in the mouse SMG.

Materials

Name	Company	Catalog Number	Comments
2-photon microscope (TCS-SP8 DIVE)	Leica, Germany
4 kDa FITC-labeled dextran	Sigma Aldrich	46944
70 kDa rhodamine B-labeled dextran	Sigma Aldrich	R9379
Blunt tissue separation nickel	Bejinghuabo Company	NZW28
Depilatory cream	Veet
Disposable sterile syringe	Zhiyu Company		1 mL
Image J software	National Institutes of Health
Insulin syringe	Becton, Dickinson and Company	0253316	1 mL
Leica Application Suite X software	Leica Microsystems
Microtubes	Axygen	MCT-150-C	1.5 mL
Phosphate buffered saline 1x	Servicebio	G4207-500
Tissue scissors	Bejinghuabo Company	M286-05
Tribromoethanol	JITIAN Bio	JT0781